Project 2-Project Summary/Abstract It is commonly assumed that major regulatory mechanisms responsible for limiting thrombus development have been identified. However, recent findings showing that TFPIa binds coagulation factor V(a) and dampens thrombin generation questions this dogma. Work from my laboratory has laid the groundwork for deciphering the mechanistic bases that may underpin how TFPIa mediates these anticoagulant effects. We think that through molecular mimicry, TFPIa uses key structural regions of the FV B-domain that remain following partial cleavage of FV during the early phases of thrombus development. These include basic (BR) and acidic (AR) regions that are key autoinhibitory elements responsible for keeping FV in an inactive procofactor state. The objectives of this proposal are to decipher these molecular processes, examine how they influence FV(a) activation and function, provide evidence its biologically relevant and identify which pool of FV (plasma or platelet-derived) contributes to these effects. A further goal is to better understand the molecular bases by which TFPIa modulate naturally occurring forms of FV including FV-East Texas and FV- Amsterdam, recently identified spliced forms of FV found in plasma that are missing a large portion of the B- domain. These new angles of thinking about FV activation, its biology, and regulation opens up several unexplored lines of experimentation. In the first aim, we will characterize the structural/functional determinants that maintain the FV procofactor state and determine whether TFPIa employs these molecular surfaces to dampen cofactor function. New biochemical and structural approaches (in collaboration with Project 1, 3 and Core B) will be pursued to provide detailed insight into these molecular interactions. In the second aim, we will investigate the biochemical properties of naturally occurring forms of FV that lack the BR and characterize their interaction with TFPIa. We hypothesize that TFPIa utilizes a different set of structural determinants to bind to different forms of FV(a) and the complex mediates its anticoagulant effects through multiple mechanisms. In the last aim, we will address the physiologic relevance of the FVAR-TFPIa interaction, determine how it may regulate thrombus development in vivo, and investigate whether this is mediated by plasma and/or platelet forms of FV. We hypothesize partially cleaved forms of FV are abundant at the site of the developing thrombus and that they are targets for TFPIa. We speculate that disruption of this regulatory mechanism results in altered kinetics of thrombus growth. We will test these innovative ideas using biochemical, kinetic, biophysical, and in vivo approaches employing a host of custom FV reagents. Knowledge gained from this proposal will move the field forward by providing an exceptional level of molecular detail on the structural and functional regulation of FV released at the site of vascular injury. Because FV/Va has such a profound impact on thrombin generation, studies in this application will have important implications in developing new ways to modulate hemostasis to control bleeding or limit thrombosis.